1. Field of the Invention
The present invention relates to methods and reagents for detecting the presence of mycobacterial nucleic acid and identifying the mycobacterial species from which a mycobacterial nucleic acid in a sample originates.
2. Description of Related Art
Mycobacteria are slow growing, acid-fast, aerobic bacilli. At least nineteen Mycobacterium species have so far been associated with disease in humans, most notably M. tuberculosis, M. bovis, and M. leprae. Some species, such as M. avium, M. intracellulare, and M. kansasii, though not normally pathogenic to healthy individuals, may cause disease in immunocompromised individuals, such as those infected with the ADS virus. In addition, several species rarely cause disease in humans but may occur in clinical specimens as saprophytes. Methods for the detection and identification of Mycobacterium species include bacterial culture, antibody detection, and, more recently, detection of rRNA by hybridization with a radioactively labelled nucleic acid probe. Each of these methods has considerable problems.
Detection by culturing the bacilli is slow, requiring up to two months, and typically requires additional biochemical testing for species identification. Antibody detection lacks specificity because of cross-reactivity between mycobacteria species and also lacks sensitivity. Furthermore, differentiation between current and past infections is difficult. Detection using radioactively labelled DNA fragments as probes that hybridize to the small subunit ribosomal RNA (16S rRNA) lacks sensitivity and still requires at least a several-day culturing period (see PCT/WO 84/02721).
The invention of the polymerase chain reaction (PCR), a method for amplifying specific sequences of nucleic acids, makes possible the rapid detection of nucleic acids present in a cell in what was previously an undetectably low quantity. Using PCR amplification, one can detect even a single copy of the target nucleic acid. Direct detection by hybridization with a sequence-specific oligonucleotide probe of a nucleic acid sequence amplified to a detectable level makes possible diagnostic tests that are specific enough to detect single nucleotide changes in sequence. However, not all primer pairs and probes are useful. The choice of primers and, hence, the region to be amplified, along with the choice of probes largely determines the specificity and sensitivity obtainable.
Amplification by the PCR hits been used in the sequencing of mycobacterial nucleic acid, detection of mycobacterial nucleic acids in a sample, and identification of mycobacteria species. Various regions of the bacterial genome have been used to detect and identify mycobacterial nucleic acids in samples. Most of these diagnostic tests were designed to detect only one or a small number of species, and limited specificity checks, if any, were performed against non-mycobacterial DNA.
Detection of a region of the gene that encodes the 65 kilodalton antigen was described in Chia et al., 1990, J. Clin. Microbiol. 28(9):1877-1880; Brisson-Noel et al., 1989, Lancet 334:1069-1071; Hackel et al., 1990, Molecular and Cellular Probes 4:205-210; Woods and Cole, 1989, FEMS Microbiology Letters 65:305-310; and Hance et al., 1989, Molecular Microbiology 3(7):843-849. No more than three sets of mycobacteria species were distinguished in any one test based on the 65 kilodalton antigen gene.
Amplification of the repetitive DNA element, IS6110, was reported in Thierry et al., 1990, J. Clin. Microbiol. 28(12):2668-2673, and Eisenach et al., 1990, J. Infectious Disease 161:977-981. Amplification of IS6110 basically serves only to test for the presence of particular species of mycobacteria, although M. tuberculosis and M. bovis can be distinguished by copy number (Plikaytis et al., 1991, Molecular and Cellular Probes 5:215-219).
The 36 kilodalton antigen of M. leprae was used in a diagnostic test in Hartskeerl et al., 1989, J. Gen. Microbiol. 135:2357-2364. Though the test was meant to be specific for M. leprae, weak to moderate hybridization to DNA from other mycobacteria was observed.
The gene sequence coding for protein antigen b was used in Sjobring et al., 1990, J. Clin. Microbiol. 28(10):2200-2204, to produce a test for M. tuberculosis/bovis based on the presence or absence of an amplified product.
A test solely for the presence of M. tuberculosis based on the gene sequence encoding the MPB 64 protein was described in Shankar et al., 1990, Lancet 335:423.
Probes constructed from cloned DNA fragments were described in Patel et al., 1990, J. Clin. Microbiol. 28(3):513-518, and Fries et al., 1990, Molecular and Cellular Probes 4:87-105. Probe specificity was obtained through a selection process rather than by sequence analysis during the probe design.
One of the regions of the mycobacterial genome that has been analyzed and targeted for use in a diagnostic test is the small subunit ribosomal RNA (16S rRNA). In Bottger, 1989, FEMS Microbiology Letters 65:171-176, the 16S rRNA genes from a variety of organisms were amplified using "universal" primers designed to amplify nucleic acid from a wide range of organisms and then directly sequenced. The phylogenetic relationship of mycobacterial species was studied by comparing 16S rRNA germ sequences in Rogall et al. 1990, J. Gen. Micro. 136:1915-1920. In Boddinghaus et al., 1990, J. Clin. Microbiol. 28(8): 1751-1759, evidence was presented regarding determinations that can be made using sequence specific oligonucleotides for amplification and hybridization to regions of the 16S rRNA sequence. A highly variable region of the 16S rRNA sequence was studied with respect to three mycobacteria species. Genus specific primers were used to amplify a region containing the variable region used for species specific probe hybridization.
The small subunit rRNA from a large number of organisms, both closely and distantly related to mycobacteria, has been studied and sequenced. A compilation of small subunit rRNA sequences from a large number of organisms is provided by Neefs et al., 1990, Nuc. Acids Res. Supplement 18:2237-2317.
There is still a need for a rapid and sensitive test to identify the presence of mycobacterial DNA and the species from which the DNA originates.